The major emphasis of this proposal is the development and assay of new inhibitors of steroid hormone biosynthesis and studies on their mechanisms of action. Information gained from structure-activity relations and specificity and mechanism of inhibition will promote our understanding of the molecular details of steroidogenic reactions and provide insight into the design of drugs for treatment of endocrine-related diseases and cancers in which steroid production plays a critical role. Initial investigations will focus on cytochrome P-450scc catalyzed conversion of cholesterol to pregnenolone. The biochemical properties of P-450scc purified from bovine adrenal cortex and human placenta will be studied. Based on our recent findings of inhibition of P-450scc by substituted benzidines, additional derivatives as well as related aryl amines will be examined to establish structure-activity correlations. Based on these findings, analogs of aminoglutethimide incorporating the most effective structural modifications will be synthesized and tested. In addition, steroid derivatives with nitrogen base substituents will be prepared as active-site directed inhibitors. The kinetics of inhibition will be studied in a reconstituted system of purified enzymes, in mitochondria from adrenal cortex, testis, ovary and placenta, and in normal and adrenal tumor cells in culture; parallel physicochemical studies on inhibitor complexes with purified P-450scc will be used to investigate the mechanism of the inhibitor-enzyme interaction. The specificity of inhibition will be investigated by assaying other steroid P-450 mono-oxygenases, 11Beta-hydroxylase and aromatase. In the long term, well characterized inhibitors would be useful for the control of normal steroid production in pregnancy or excess production associated with pituitary or adrenal tumors and as antineoplastic agents in blocking the androgen or estrogen synthesis required for the growth of certain steroid-dependent cancers.